Genome Whole Associated Analysis And Molecular Identification And Functional Analysis Of Genes Regulating Heading Date In Common Wheat

Heading is one of the most important traits in wheat as it affects growth and development and adaptation and yield potential,which was modulated by vernalization and photoperiod response genes mostly,there are many other genes or loci affecting heading.In the present study,163 winter wheat cultivars and advanced lines from the Yellow and Huai valleys,205 germplasms collected from China,and two RIL populations were used to this research,the method of GWAS and QTL mapping and the technology of homology cloning,RT-PCR and transgenic were used to discover the new loci affecting heading date and identify alleles of vernalization and photoperiod genes.The main results obtained in this study are summarized as below:1.GWAS analysis identified 306 significant SNPs associating heading and flowering dates using 90 K assay and mapping population with 13 environments,which mainly distributed on chromosomes 2A,2B,2D,5A,5B,6A,6D,7A,and 7B.Among these,13 stable SNPs were significantly associated with heading or flowering dates in more than four environments.Further functional analysis suggested that ten significant SNPs of them related to transport protein,disease stress protein and cytochrome gene through NCBI blasting.2.A novel allele was discovered at Vrn-D1 locus by identifying the polymorphisms of vernalization and photoperiod genes,and named Vrn-D1 c,of which a175 bp insertion in the promoter region compared to recessive allele vrn-D1.Functional analysis of promoter showed that there were five cis-elements?Box II-like,3-AF1 binding site,TC-rich repeats,Box-W1 and CAT-box?in the insertion,which possiblyaffect the function of Vrn-D1 c allele.Quantitative real-time PCR?q RT-PCR?showed that cultivar with Vrn-D1 c allele possessed significantly higher expression level of Vrn-D1 c gene than cultivar with recessive allele vrn-D1,indicating that the 175-bp insertion of Vrn-D1 c contributed to increased expression level of Vrn-D1 gene and caused to earlier head and flower under the condition of non-vernalization treatment.3.There were 13 allele totally founded?vrn-A1,Vrn-A1 a,Vrn-A1 b,vrn-B1,Vrn-B1 a,Vrn-B1 b,vrn-D1,Vrn-D1 a,Vrn-D1 b,Vrn-D1 c,vrn-D3,Vrn-D3 a,and Vrn-D3b?at four loci?vrn-A1,vrn-B1,vrn-D1,and vrn-B3?and recessive allele was predominant in these four loci.There were 19 allelic combinations in all cultivars according to this four loci,among which vrn-A1/vrn-B1/vrn-D1/vrn-B3 has the highest frequency with 43.9%,the cultivars possessing this combination had much longer heading and flowering dates.4.Polymorphisms of Ppd-D1 and Ppd-B1 genes were identified using specific primers.Results showed that there were 3 polymorphisms at Ppd-D1 locus,resulting in six haplotypes?Ppd-D1_Hapl_I-Ppd-D1_Hapl_XI?,among which Ppd-D1_Hapl_IX and Ppd-D1_Hapl_X were both discovered at the first time in wheat.Besides,Ppd-D1_Hapl-I was predominant in all cultivars with 90.24%.Results showed that there were 3 Ppd-B1 a alleles at Ppd-B1 locus,resulting in eight haplotypes?Ppd-B1_Hapl-I-Ppd-B1_Hapl-??,among these,cultivars with Ppd-B1_Hapl-VI had the shortest heading and flowering dates,179 d and 184 d,respectively;whereas cultivars with Ppd-B1_Hapl-I had the longest heading time and flowering dates,184 d and 190 d,respectively.5.We proposed an linear regression equation based on 7 vernalization and photoperiod loci and new SNPs identifying by GWAS associated with with heading and flowering dates under different environmentsin wheat cultivars among three populations,which was optimized by eliminating not significantly factors,and found that Vrn-B1,Vrn-D1,Ppd-D1,RAC875_c41145₁89,Excalibur_c60164₁37,and RAC875_c50422₂99 were affecting heading and flowering dates.Further evaluatedthe effects of these loci by path coefficients?pi?,which indicated that the Ppd-D1 gene plays the most important role in heading and flowering dates in Chinese wheat.Using90 K assay and Vrn-B1,Vrn-D1 and Ppd-D1 loci with mapping population to GWAS analysis again,results showed that Ppd-D1 gene significantly associated in several environments.Linkage analysis using a RIL population of PC indicated that a major LOD at the Ppd-D1 locus not only controlling heading and flowering dates but also significantly associated with peduncle length,spike length,fertile spikelets,cold resistance,and tiller number.6.There was a major QTL locus affecting heading date by QTL mapping in RIL population of UP,located between SSR marker barc154-7D and barc126 with distance of 3.0 c M and LOD of 5.30 to 12.8 and PVE of 8.34% to 25.94%.Transcriptome analysis showed that there was a gene located in this interval,which is highly homology to FT gene in Arabidopsis and Vrn-B3 gene in wheat and named Vrn-D3 gene.Sequencing results showed that there was a frameshift mutant resulting in extended to60 amino acids in PI 610750 of Vrn-D3 gene,and this allele was named Vrn-D3 b and another allele was Vrn-D3 a.Subcellular localization results showed that Vrn-D3 gene expressed in the nuclear.Transgenic results showed that flowering and maturity dates of transgenic lines were earlier compared to control under different vernalization treatments and photoperiod treatment in Arabidopsis.Transgenic results also showed that heading and maturity dates of transgenic lines were earlier compared to control under different vernalization treatments in wheat,and there were difference with architecture,plant height and other traits between transgenic and control lines.Summarize the above results,this research identified many SNPs through GWAS analysis with heading and flowering dates,and some stable SNPs to further analyse phenotype and function annotation;found a novel Vrn-D1 allele by identifying the polymorphsim of vernalization gene,and analysed its sequence and function;identified many alleles or haplotypes and associated with heading and flowering dates by PCR amplifying with vernalizaton and photoperiod genes;proposed an optimal linearregression equation and showed that Ppd-D1 gene plays the most important role in heading and flowering dates in Chinese wheat;identified a novel allele of Vrn-D3 gene using RIL population combine with transcriptome analysis and homology gene cloning,and validated function by transgenic of this allele.